Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ~12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH.

In conditions of facilitated synaptic release, CA3/CA1 synapses generate anomalously slow NMDA receptor-mediated EPSCs (EPSC(NMDA)). Such a time course has been attributed to the cooperation of synapses through glutamate spillover. Imitating a natural pattern of activity, we have applied short bursts (2-7 stimuli) of high-frequency stimulation and observed a spike-to-spike slow-down of the EPSC(NMDA) kinetics, which accompanied synaptic facilitation. It was found that the early component of the EPSC(NMDA) and the burst-induced late component of the EPSC(NMDA) have distinct pharmacological properties. The competitive NMDA antagonist R-(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (D-CPP), which has higher affinity to NR2A than to NR2B subunits and lowest affinity at NR2D subunits, significantly slowed down the decay rate of the afterburst EPSC while leaving the kinetics of the control current unaffected. In contrast, ifenprodil, a highly selective NR2B antagonist, and [+/-]-cis-1-[phenanthren-2yl-carbonyl]piperazine-2,3-dicarboxylic acid (PPDA), a competitive antagonist that is moderately selective for NR2D subunits, more strongly inhibited the late component of the afterburst EPSC(NMDA). The receptors formed by NR2B and (especially) NR2D subunits are known to have higher agonist sensitivity and much slower deactivation kinetics than NR2A-containing receptors. Furthermore, NR2B is preferentially and NR2D is exclusively located on extrasynaptic membranes. As the density of active synapses increases, the confluence of released glutamate makes EPSC decay much longer by activating more extrasynaptic NR2B- and NR2D-subunit-containing receptors. Long-term potentiation (LTP) induced by successive rounds of burst stimulation is accompanied by a long-term increase in the contribution of extrasynaptic receptors in the afterburst EPSC(NMDA.)

piperazine-2,3-dicarboxylic acid (PBPD) is a moderate affinity, competitive N-methyl-D-aspartate (NMDA) receptor antagonist with an atypical pattern of selectivity among NMDA receptor 2 subunit (NR2) subunits. We now describe the activity of several derivatives of PBPD tested at both rat brain NMDA receptors using L-[ 3 H]-glutamate binding assays and at recombinant receptors expressed in Xenopus oocytes. 2 Substituting various branched ring structures for the biphenyl group of PBPD reduced NMDA receptor activity. However, substituting linearly arranged ring structures -fluorenone or phenanthrene groups -retained or enhanced activity. 3 Relative to PBPD, the phenanthrene derivative (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) displayed a 30-to 78-fold increase in affinity for native NMDA receptors. At recombinant receptors, PPDA displayed a 16-fold (NR2B) to 94-fold (NR2C) increase in affinity over PBPD. 4 Replacement of the biphenyl group of PBPD with a 9-oxofluorene ring system resulted in small changes in receptor affinity and subtype selectivity. 5 2 0 -Bromo substitution on the biphenyl group of PBPD reduced antagonist affinity 3-to 5-fold at NR2A-, NR2B-and NR2D-containing receptors, but had little effect on NR2C-containing receptors. In contrast, 4 0 -fluoro substitution of the biphenyl ring of PBPD selectively increased NR2A affinity. 6 The aromatic rings of PBPD and PPDA increase antagonist affinity and appear to interact with a region of the NMDA receptor displaying subunit heterogeneity. PPDA is the most potent and selective NR2C/NR2D-preferring antagonist yet reported and thus may be useful in defining NR2C/ NR2D function and developing related antagonists with improved NMDA receptor subtype selectivity.

Mutations in FGFR1/FGF8/PROKR2 contributed to 7.8% of our patients with CPHD/SOD. These data suggest a significant genetic overlap between conditions affecting the development of anterior midline in the human forebrain.